Methods of producing phosphitylated compounds

ABSTRACT

Provided are methods of producing phosphitylated compounds, including 3′-O-phosphoramidites, comprising the step of reacting a hydroxyl-containing compound with a phosphitylating agent in the presence of a phosphitylation activator selected from the group consisting of: (1) acid-base complexes derived from an amine base of Formula I  
                 
 
     wherein R, R 1 , and R 2  are independently C 1 -C 10  alkyl, C 1 -C 10  cycloalkyl, C 1 -C 10  aryl, C 1 -C 10  aralkyl, C 1 -C 10  heteroalkyl, or C 1 -C 10  heteroaryl; (2) acid-base complexes derived from an amine base of Formula II  
                 
 
     wherein R 3 , R 4 , R 5 , R 6 , and R 7  are independently hydrogen, C 1 -C 10  alkyl, C 1 -C 10  cycloalkyl, C 1 -C 10  aryl, C 1 -C 10  aralkyl, C 1 -C 10  heteroalkyl, or C 1 -C 10  heteroaryl, and at least one of R 3 , R 4 , R 5  R 6 , and R 7  is not hydrogen.; (3) acid-base complexes derived from a diazabicyclo amine base; (4) zwitterionic amine complexes; and (5) combinations of two or more thereof, to produce a phosphitylated compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. ProvisionalApplication Serial No. 60/359,124 which was filed with the United StatesPatent and Trademark Office on Feb. 22, 2002, and U.S. ProvisionalApplication Serial No. 60/362,320 which was filed with the United StatesPatent and Trademark Office on Mar. 7, 2002. Both of the aforementionedprovisional applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention is directed to methods of producingphosphitylated compounds by reacting a hydroxyl-containing compound witha phosphitylating agent in the presence of a phosphitylation activator.

BACKGROUND

[0003] The production of phosphitylated compounds via the reaction ofhydroxyl-containing compounds with phosphine reagents is atransformation that has found utility in the synthesis of a wide rangeof useful compounds. For example, applicants have recognized that such atransformation is useful in the synthesis of 3′-O-phosphoramidites from5′-O-protected nucleosides, as shown generally in Scheme 1:

[0004] wherein, for example, X is hydrogen, alkoxy,-O-tert-butyldimethyl silyl (OTBDMS), —O-methoxy methyl (OMOM),2′-O-methoxyethyl (2′-O-MOE), and the like; R′ is DMT, dimethoxytrityl,oligonucleotides and analogs thereof, and the like; R″ is alkyl, such asmethyl and the like, or alkoxy, such as 2-cyanoethyl and the like; R′″is diisopropylamine and the like; and B is moiety derived from adenine,cytosine, guanine, thymine, or uracil.

[0005] Phosphoramidites of the type formed via Scheme 1 can beadvantageously coupled to prepare oligonucleotides, see for example U.S.Pat. No. 4,725, 677 and Mellor, Thomas, “Synthesis of analogues ofoligonuclotides”, J. Chem Soc., Perkin Trans. 1, 1998, 747-757 (both ofwhich are incorporated herein by reference), which have a risingimportance in the field of therapeutic and diagnostic applicationsincluding, for example, antisense drugs (as described in Crooke, S. T.Handbook of Experimental Pharmacology: Antisense Research andApplication; Springer-Verlag, Berlin, (1998), incorporated herein byreference). To supply the growing demand for these oligonucleotides,there is a desire to improve the synthesis of nucleosidicphosphoramidites on a commercial scale (Noe, Kaufhold, New Trends inSynthetic Medicinal Chemistry, Wiley-VCh Weinheim, 2000, 261,incorporated herein by reference).

[0006] However, applicants have come to appreciate that conventionalmethods for preparing phosphitylated compounds, such as3′-O-phosphoramidites, from hydroxyl-containing compounds aredisadvantageous for several reasons. One disadvantage associated withmany conventional methods is the required use of costly and/or hazardousactivating agents/compounds. For example, in Beaucage and Carruthers,Tetrahedron Lett. 1981, 22, 1859 (incorporated herein by reference),1H-Tetrazole is recommended as the most versatile phosphitylationactivator. However, such an activator/reagent is both expensive andhazardous. (See, for example, Stull, Fundamentals of Fire and Explosion,AlChE Monograph Series, No. 10, New York, 1977, Vol. 73, 22,incorporated herein by reference). Due to the explosive nature of thenitrogen-rich heterocycle, special safety precautions are required forthe handling of such compositions. A less hazardous compound,4,5-Dicyanoimidazole, has been shown to be useful in the production ofcertain nucleosidic phosphoramidites. Unfortunately, this compound isvery expensive, and, in fact, tends to be prohibitively expensive withregard to its use in industrial processes. Phosphitylation activatorsderived from unsubstituted pyridine are disclosed, for example, inGryaznov, Letsinger, J. Am. Chem. Soc. 1991, 113, 5876; Gryaznov,Letsinger, Nucleic Acids Res. 1992, 20, 1879; Beier, Pfleiderer,Helvetica Chimica Acta, 1999, 82, 879; Sanghvi, et al., Organic ProcessResearch and Development 2000, 4, 175; and U.S. Pat. No. 6,274,725,issued to Sanghvi et al., all of which are incorporated herein byreference. However, these salts tend to be toxic and highly watersoluble. Accordingly, cost-intensive waste water treatment equipmentmust be installed in systems using such activators.

[0007] Another disadvantage associated with many conventional methodsfor preparing phosphitylated compounds is the use of dichloromethane asthe preferred solvent. Because dichloromethane tends to beenvironmentally unfriendly, relatively costly waste treatment equipmentis required for use in conjunction with methods involvingdichloromethane as solvent.

[0008] One potential approach to avoid at least some of theaforementioned disadvantages is in situ preparation of nucleosidicphosphoramidites without an additional activation step, as described,for example, by Zhang et al., U.S. Pat. No. 6,340,749 BI, for immediateuse of the resulting solution on the solid support synthesizer.Unfortunately, such methods tend to be relatively inefficient, and thephosphoramidite solutions obtained via such methods tend to be unstableand unsuitable for storage.

[0009] Accordingly, applicants have recognized the need for new methodsof producing phosphitylated compounds which avoid the disadvantagesassociated with conventional methods.

DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

[0010] The present invention overcomes the aforementioned shortcomingsby providing efficient methods of producing a wide variety ofphosphitylated compounds, which methods tend to be less hazardous andless costly than conventional methods. Specifically, applicants havediscovered that certain acid-base complexes and zwitterionic complexesderived from relatively sterically-hindered amine bases can be used togreat advantage as phosphitylation activators in methods of preparingphosphitylated compounds from hydroxyl-containing starting materials. Asused herein, the term “phosphitylated compound” refers generally to acompound containing an oxygen-phosphorus bond formed via the reaction ofa hydroxyl-containing compound with a phosphitylating agent. Applicantshave discovered that acid-base and zwitterionic complexes of the presentinvention tend to be both less toxic and less water soluble thanconventional activators.

[0011] In addition, applicants have discovered unexpectedly that, inmany embodiments, the methods of the present invention allow for theproduction of phosphitylated compounds in yields at least as high, andin certain cases, higher than those achieved via prior art processesdespite the fact that the acid-base and zwitterionic activator complexesof the present invention tend to be more sterically-hindered, and lessnucleophilic, than activators used conventionally. Although applicantsdo not wish to be bound by or to any particular theory of operation, itis believed that the mechanism of activating a phosphitylating agent foruse in the production of phosphitylated compounds involves thenucleophilic displacement of a leaving group on the phosphitylatingagent by the activator. For example, Berner et al. Nucleic Acids Res.1989, 17, 853 and Dahl, B., et al., Nucleic Acids Res. 1987, 15, 1729describe the proposed mechanism of activation of the bis-reagent2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite with aless-sterically hindered amine activators (such as tetrazole) asinvolving the displacement of a diisopropylamine leaving group by thenucleophilic amine, prior to the reaction of the activated bis-reagentagent with a hydroxyl-containing compound.

[0012] In light of this, it would be expected that the presentactivators comprising amines that are relatively, and in many casessignificantly, more sterically-hindered than prior art amine activators,such as tetrazole and pyridine activators, would be less efficient indisplacing leaving groups on phosphitylating agents, and therefor lessefficient in activating such agents to produce phosphitylated compounds.Nevertheless, as noted above, applicants have found, surprisingly, thatsignificantly sterically-hindered activators of the present inventionallow for the production of phosphitylated products in yields as good oreven better than conventional activators, such as the salts ofunsubstituted pyridine. Without intending to be bound by or to anyparticular theory of operation, subsequent investigation into thediscovered unexpected results has lead applicants to believe that thesurprisingly high yields associated with present activators may be due,at least in part, to reduced side reactions of the activator withreactive moieties of certain hydroxyl-containing starting materials(e.g. the lactam unit of guanosine compounds as discussed by Nielsen etal. Nucleic Acids Res. 1986, 14, 7391.) Accordingly, the present methodsallow for the production of phosphitylated compounds in yields as goodas, and often better, than conventional methods while also avoiding manyof the disadvantages associated with such conventional methods.

[0013] According to certain embodiments, the methods of the presentinvention comprise the step of reacting a hydroxyl-containing compoundwith a phosphitylating agent in the presence of a phosphitylationactivator selected from the group consisting of: acid-base complexesderived from amines of Formula I or Formula II, described below,acid-base complexes derived from diazabicyclo amine compounds,zwitterionic amine complexes, and combinations of two or more thereof,to produce a phosphitylated compound.

[0014] Phosphitylation Activator

[0015] As used herein the term “phosphitylation activator” refersgenerally to a compound that promotes the reaction of ahydroxyl-containing compound with a phosphitylating agent to produce aphosphitylated compound according to the present invention. Applicantshave discovered that a wide range of acid-base complexes and zwitterioncomplexes can be used to great advantage as phosphitylation activators.

[0016] A. Acid-Base Complexes

[0017] The complexes of acids and bases of the present invention areformed by introducing at least one amine base of Formula I or FormulaII, described below, or a diazabicyclo amine base, to at least one acidto form an acid-base complex.

[0018] wherein R, R¹, and R² are independently alkyl, cycloalkyl, aryl,aralkyl, heteroalkyl, or heteroaryl, each having from about 1 to about10 carbons (C₁-C₁₀).

[0019] wherein R³, R⁴, R⁵, R⁶, and R⁷ are independently hydrogen, C₁-C₁₀alkyl, C₁-C₁₀ cycloalkyl, C₁-C₁₀ aryl, C₁-C₁₀ aralkyl, C₁-C₁₀heteroalkyl, or C₁-C₁₀ heteroaryl, wherein at least one of R³, R⁴, R⁵,R⁶, and R⁷ is not hydrogen.

[0020] R, R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ as C₁ to C₁₀ alkyl groups maybe straight chain or branched moieties, for example, substituted orunsubstituted: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl,2-ethylhexyl, nonyl, decyl and the like. Any of these groups may besubstituted with halogen, hydroxyl, alkoxy, aryloxy, alkyl, fluoroalkyl,arylalkyl groups, and the like.

[0021] R, R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ as C₁ to C ₁₀ cycloalkyls maybe, for example, substituted or unsubstituted: cyclopropyl, cyclobutyl,cyclopentyl, methylcyclopentyl, cyclohexyl, methylcyclohexyl,dimethylcyclohexyl, cycloheptyl, cyclooctyl, and the like. Any of thesegroups may be substituted with, for example, halogen, hydroxyl, alkoxy,aryloxy, alkyl, fluoroalkyl, arylalkyl groups, and the like.

[0022] R, R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ as C₁ to C₁₀ aryls may be, forexample, substituted or unsubstituted: phenyl, o-tolyl, m-tolyl,p-tolyl, o-xylyl, m-xylyl, p-xylyl, alpha-naphthyl, beta naphthyl, andthe like. Any of these groups may be substituted with, for example,halogen, hydroxyl, aryloxy, alkyl, fluoroalkyl, arylalkyl groups, andthe like.

[0023] R, R¹, R², R³, R⁴, R⁵, R⁶, and R⁷ as C₁ to C₁₀ aralkyls may be,for example, substituted or unsubstituted: benzyl, 4-methylbenzyl,o-methylbenzyl, p-methylbenzyl, diphenylmethyl, 2-phenylethyl,2-phenylpropyl, 3-phenylpropyl, and the like. Any of these groups may besubstituted with, for example, halogen, hydroxyl, aryloxy, alkyl,fluoroalkyl, arylalkyl groups, and the like.

[0024] Any two adjacent R, R¹, and R², or R³, R⁴, R⁵, R⁶, and R⁷ groupsin Formulae I and II, respectively, may be connected to form anaromatic, non-aromatic, or heterocyclic ring.

[0025] Examples of amine bases of Formula I suitable for use in thepresent methods include: trialkylamines, such as, diisopropylethylamine(i.e. Hünig's Base), tripropylamine, triethylamine, trimethylamine,diethylmethylamine, N-methylmorpholine (NMM) and the like; tertiarydiamines, such as, tetramethylethylendiamine (TMEDA); polyamines andpolymer bound alkylamines; triarylamines, such as, triphenylamine, andthe like; triaralkylamines, such as, tribenzylamine, and the like; othertrisubstituted amines, such as dimethylaniline; and the like. Certainpreferred amine bases of Formula I include Hünig's Base, and the like.

[0026] Examples of amine bases of Formula II suitable for use in thepresent methods include: dimethylaminopyridine (DMAP),4-dimethylaminopyridine, and other substituted pyridines, such as,monoalkylpyridines, including methylpyridine, 2-picoline, 3-picoline,dialkylpyridines, including dimethylpyridine, 2,6-lutidine,trialkylpyridines, including trimethylpyridine, 2,4,6-collidine,syn-collidine, tetraalkylpyridines, including tetramethylpyridine, andpentaalkylpyridines, including pentamethylpyridine, and the like.Certain preferred bases of Formula II include 2-picoline, syn-collidine,and the like.

[0027] Examples of diazabicyclo amine bases suitable for use in thepresent methods include 1,5-diazabicyclo[4.3.0]non-5-ene (DBN);1,8-diazabicyclo[5.4.0]undec-7-ene (DBU); 1,1,3,3-tetramethylguanidine;and the like.

[0028] Any of a wide range of acids may be combined with one or morebases of the present invention to form an acid-base complex of thepresent invention. Suitable acids include: acetic acid derivaties, suchas, trifluoroacetic acid (TFA), dichloroacetic acid, and the like;sulfonic acids, such as, methane sulfonic acid, trifluoromethanesulfonic acid, 4-pyridiniumethylene sulfonic acid, and the like,non-aqueous hydrogen halide acids, such as, non-aqueous hydrogenchloride, non-aqueous hydrogen bromide, non-aqueous hydrogen iodide, andthe like; and HBF₄. In certain preferred embodiments, the acid used inthe present invention is trifluoroacetic acid.

[0029] Certain preferred acid-base complexes of the present inventioninclude complexes of: trifluoroacetic acid and Hünig's Base;trifluoroacetic acid and 2-picoline; and trifluoroacetic acid andsyn-collidine. An especially preferred acid-base complex is a complex oftrifluoroacetic acid and Hünig's Base.

[0030] Any of a wide range of known methods for forming acid-basecomplexes can be adapted for use in making acid-base complexes accordingto the present invention. For example, in certain embodiments, thepresent acid-base complexes are produced by introducing at least oneacid to at least one base to form the complex.

[0031] The acids and bases of the present invention may be introduced inthe presence or absence of solvent to form the present complexes. Inembodiments including the presence of solvent, either or both of theacid and/or base may be first dissolved in solvent to form an acidsolution and/or base solution, prior to contacting the acid and base toform the complex. The solvent used in forming an acid-base complex ofthe present invention may be the same or different from any optionalsolvent used in the phosphitylation reaction of the present invention.Solvents suitable for use in making an acid-base complex according tothe present invention include non-polar and polar, aprotic solvents.Examples of suitable non-polar and polar, aprotic solvents include:acetates, such as, methyl acetate, ethyl acetate, propyl acetate,isopropyl acetate, n-butyl acetate, isobutyl acetate, and the like;ethers, such as, tetrahydrofuran (THF), methyl tert-butyl ether (MTBE),and the like; aromatic solvents, such as, toluene, chlorobenzene, andthe like; dichloromethane; acetonitrile; N-methyl-2-pyrrolidone (NMP);N,N-dimethylformamide (DMF) and combinations of two or more of these. Incertain embodiments, the base of the acid-base complex, or the complexitself may act as solvent.

[0032] Any suitable amounts of acid and base can be used in preparingthe present acid-base complexes. In general, sufficient acid and baseshould be used such that the acid and base components are present in thecomplex in a molar ration of about 1:1. In certain embodiments, fromabout 0.9 to about 1.5 equivalents of base and from about 0.9 to about1.5 equivalents of acid are used. Preferably, from about 0.9 to about1.3 equivalents of base and from about 0.9 to about 1.1 equivalents ofacid are used, and even more preferably from about 1.0 to about 1.3equivalents of base and from about 1.0 to about 1.05 equivalents of acidare used. In certain especially preferred embodiments, about 1.3equivalents of base and about 1.05 equivalents of acid are used. Unlessotherwise indicated, all equivalents are molar equivalents.

[0033] The acid-base complexes of the present invention may be formed insitu in the phosphitylation reactions of the present invention, or maybe formed separately therefrom. In embodiments wherein the complex isformed in situ, the complex may be formed in the reaction mixture priorto adding either the phosphine and/or hydroxyl-containing compound.Alternatively, the complex may be prepared after introduction of boththe phosphine and/or hydroxyl-containing compounds to the reactionmixture by a subsequent addition of the acid and/or base of the complex.

[0034] Any suitable reaction conditions may be used to form thecomplexes of the present invention. For example, in certain embodiments,the acids and bases of the present invention are mixed at a temperatureof from about 0° C. to about 100° C. Preferably, the acids and bases aremixed at a temperature of from about 10° C. to about 60° C., and morepreferably from about 15° C. to about 40° C.

[0035] B. Zwitterion Complexes

[0036] As used herein, the term “zwitterion complex” refers generally toa complex ion having a cation and an anion in the same molecule (i.e. aninternal salt), as is known in the art. Applicants have discoveredunexpectedly that such zwitterion complexes are suitable for use asphosphitylation activators according to the present invention.

[0037] Any of a wide range zwitterionic compounds/internal salts aresuitable for use according to the present invention. Examples ofsuitable zwitterionic compounds include sulfonic acid compouds, such as,pyridineethansulfonic acid, and the like.

[0038] Phosphitylating Agent

[0039] As used herein, the term “phosphitylating agent” refers generallyto any reagent compound capable of reacting with a hydroxyl-containingcompound in the presence of a phosphitylation activator to form a bondbetween the oxygen atom of a hydroxyl group of the hydroxyl-containingcompound and a phosphorus atom of the phosphitylating agent to form aphosphitylated compound. Any of a wide range of compounds are suitablefor use as phosphitylating agents according to the present invention.Suitable compounds include, for example, phosphines, such asbis-substituted phosphines, including,alkoxy-bis(dialkylamino)phosphines, such asbis-diisopropylamino-2-cyanoethoxyphosphine;dialkoxy(dialkylamino)phosphines; alkoxy-alkyl(dialkylamino)phosphines,bis(N,N-diisopropylamino)-2-methyltrifluoroacetylaminoethoxyphosphine;bis(N,N-diisopropylamino)-2-diphenyl-methylsilylethoxyphosphine;(allyloxy)bis(N,N-dimethylamino)-phosphine; and the like; as well as,phosphoramidites, such as,hydroxyl-protected-N,N,N′,N′-phosphoramidites, including,2-cyanoethyl-N,N,N′,N-tetraisopropylphosphorodiamidite;methoxy-N,N,N′,N′-tetraisopropylphosphorodiamidite;methyl-N,N,N′,N′-tetraisopropylphosphorodiamidite, and the like, and3′-O-phosphoramidites, such as,5′-O-Dimethoxytrityl-2′-deoxyAdenosine(N⁶-Benzoyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,5′-O-Dimethoxytrityl-2′-(N⁴-Benzoyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,5′-O-Dimethoxytrityl-2′-deoxyGuanosine(N²-isobutyroyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,5′-O-Dimethoxytrityl-thymidine-3′-NN-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,and the like; and mixtures of two or more thereof. Preferredphosphitylating agents includehydroxyl-protected-N,N,N′,N′-phosphoramidites, such as,2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite,methoxy-N,N,N′,N′-tetraisopropylphosphorodiamidite,5′-O-Dimethoxytrityl-2′-deoxyAdenosine(N⁶-Benzoyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,5′-O-Dimethoxytrityl-2′-(N⁴-Benzoyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,5′-O-Dimethoxytrityl-2′-deoxyGuanosine(N²-isobutyroyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,and5′-O-Dimethoxytrityl-thymidine-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite.In a particularly preferred embodiment, the phosphitylating agent is2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite.

[0040] Hydroxyl-Containing Compounds

[0041] As used herein, the term “hydroxyl-containing compound” refersgenerally to a compound containing at least one hydroxyl group, which iscapable of reacting with a phosphitylating agent in the presence of aphosphitylation activator to form a bond between an oxygen atom of atleast one hydroxyl group of the hydroxyl-containing compound and aphosphorus atom of the phosphitylating agent to form a phosphitylatedcompound. In general, any compound comprising at least one hydroxylgroup which is capable of reacting with a phosphitylating agent in thepresence of a phosphitylation activator to form a bond between an oxygenatom of at least one hydroxyl group of the hydroxyl-containing compoundand a phosphorus atom of the phosphitylating agent to form aphosphitylated compound is suitable for use as hydroxyl-containingcompounds according to the present invention. In certain preferredembodiments, the hydroxyl containing compounds of the present inventioninclude any natural and/or non-natural nucleosides, including DNA and/orRNA nucleosides, including Linked Nucleic Acid (LNA) derivatives andnucleosides substituted with additional groups, e.g. halogenesubstituents, Detector-containing nucleosides, including Biotin- orFluorescein-linked compounds; Effector-containing compounds with ligandsenhancing antisense action; as well as oligomeric structures derivedfrom two or more of these. Examples of suitable DNA and RNA nucleosidesinclude protected nucleosides, such as 5′-O-protected nucleosides (withor without additional N-protection, such as protection via benzoyl,isobutyryl, tert-butylphenoxyacetyl “TAC”, and the like), including5′-O-protected nucleosides of Adenosine, Cytidine, Guanosine, Thymidine,deoxyAdenosine, deoxyCytidine, and deoxyGuanosine;5′-O-protected-2′-protected nucleosides, (with or without additionalN-protection), including 5′-O-protected-2′-protected nucleosides ofAdenosine, Cytidine, Guanosine, and Uridine (wherein preferred2′-protecting groups include t-butyldimethylsilyl, methoxymethyl (MOM),methoxyethyl (MOE) and alkoxy, such as, methoxy, groups), as well as3′-O-protected nucleosides of Adenosine, Cytidine, Guanosine, Thymidine,Uridine, deoxyAdenosine, deoxyCytidine, and deoxyGuanosine, (with orwithout additional N-protection) and oligomeric structures derivedtherefrom.

[0042] Reaction Solvent and Conditions

[0043] The present methods may be adapted for use as batch or continuousprocesses.

[0044] According to certain embodiments, the reaction step of thepresent phosphitylation methods further comprises a solvent. The solventused in the phosphitylation reactions of the present invention may bethe same or different from any optional solvent used in forming anacid-base complex of the present invention. Solvents suitable for use inthe phosphitylation reaction according to the present invention includenon-polar and polar, aprotic solvents. Examples of suitable non-polarand polar, aprotic solvents include: acetates, such as, methyl acetate,ethyl acetate, propyl acetate, isopropyl acetate, n-butyl acetate,isobutyl acetate, and the like; ethers, such as, THF, MTBE, and thelike; aromatic solvents, such as, toluene, chlorobenzene, and the like;dichloromethane; acetonitrile; NMP; DMF and combinations of two or moreof these. In certain embodiments, the base of the acid-base complex, orthe complex itself may act as solvent.

[0045] Any suitable amounts of hydroxyl-containing compound andphosphitylating agent can be used in the methods of the presentinvention. In certain embodiments, from about 0.9 to about 1.5equivalents of hydroxyl-containing and from about 0.9 to about 1.5equivalents of phosphitylating agent are used. Preferably, from about0.9 to about 1.1 equivalents of hydroxyl-containing compound and fromabout 0.9 to about 1.3 equivalents of phosphitylating agent are used,and even more preferably from about 0.9 to about 1.1 equivalents ofhydroxyl-containing compound and from about 1.0 to about 1.3 equivalentsof phosphitylating agent are used. In certain especially preferredembodiments, about 1.0 equivalents of hydroxyl-containing compound andabout 1.1 equivalents of phosphitylating agent are used. Unlessotherwise indicated, all equivalents are molar equivalents.

[0046] Any suitable reaction conditions, including conditions disclosedin any of the documents incorporated herein by reference, may be used inthe phosphitylation reactions of the present invention. In certainembodiments, the phosphitylation reaction is conducted at a temperatureof from about 0° C. to about 100° C. Preferably, the phosphitylationreaction is conducted at a temperature of from about 0° C. to about 40°C., and more preferably at about 20° C.

[0047] The phosphitylated compounds prepared via the present methods maybe purified via any suitable methods known in the art. For example,aqueous washes, drying, concentrating under reduced pressure,chromatography, distillation, crystallization, precipitation and thelike may be used.

[0048] According to certain preferred embodiments, applicants havediscovered that relatively-highly pure phosphitylated compounds, suchas, 3′-O-phosphoramidites, can be obtained by precipitating thephosphitylated compound in solution. In certain preferred embodiments,the precipitation methods of the present invention comprise providing acompound solution comprising a phosphitylated compound to beprecipitated and a solvent, and contacting said compound solution with aprecipitation solvent to precipitate the phosphitylated compound.

[0049] Any of a wide range of suitable solvents can be used in thecompound solutions according to the present invention. Examples ofsuitable compound solution solvents include: toluene, xylene,methylacetate, ethylacetate, propylacetate, butylacetate, combinationsof two or more thereof, and the like. Preferred compound solutionsolvents include toluene and the like.

[0050] The compound solutions can be provided via any of a wide range ofmethods according to the present invention. In certain preferredembodiments, the compound solution is provided as the product of aphosphitylation reaction of the present invention. Such solution may beobtained directly from the phosphitylation reaction or may be providedby purifying a reaction product and solvating such purified product.Alternatively, a phosphitylated compound obtained via a source otherthan a reaction of the present invention can be dissolved in a compoundsolution solvent to provide a compound solution according to the presentinvention.

[0051] Any suitable precipitation solvent can be used to precipitatephosphitylated compounds according to the present invention includingalkanes, such as, petroleum ether, pentane, hexane, isohexane, heptane,isooctane, and the like, and mixtures of two or more thereof. Preferredprecipitation solvents include petroleum ether, hexane, mixtures of twoor more thereof, and the like.

[0052] In certain preferred embodiments, one or more additives can beadded to the precipitation solvent to influence the structure of theprecipitate in methods of the present invention. Examples of suitableadditives include, for example, triethylamine, and the like. Anysuitable amount of additive can be added to a precipitation solventaccording to the present invention. In certain preferred embodiments,from about 0 to about 10% by weight, based on the total weight ofprecipitation solvent and additive, of additive is used, preferably fromabout 0 to about 5% is used.

[0053] Any suitable ratio of compound solution to precipitationsolvent/additives can be used according to the present methods. Incertain preferred embodiments, the compound solution is added to about 5to about 25 equivalents by weight, preferably about 20 to about 25equivalents, of precipitation solvent or precipitation solvent andadditive (if present).

[0054] The precipitation can be conducted under any suitable conditionsand using any suitable laboratory equipment. Preferably theprecipitation is conducted under an inert gas atmosphere, such asnitrogen, argon, or the like. Any suitable temperature can be used, forexample, from about −20° C. to about 40° C. Preferably, precipitation isconducted at a temperature of from about 0° C. to about 30° C., and morepreferably, from about 5° C. to about 25° C. Any suitable vessels can beused for precipitation. In certain preferred embodiments, stainlesssteel vessels are used.

[0055] Automated Oligonucleotide Synthesis

[0056] As will be recognized by those of skill in the art,oligonucleotides may be synthesized from hydroxyl-containing compoundscomprising nucleosides and/or oligomers derived therefrom according tothe present methods, not only via the batch and/or continuous processesdescribed above, but also using automated oligonucleotide synthesistechniques, as described, for example, in Applied BioSystems User'sManual for Models 392 and 394 DNA/RNA Synthesizers; Section 6 Chemistryfor Automated DNA/RNA Synthesis (March 1994) and M. J. Gait,“Oligonucleotide Synthesis, A Practical Approach”, IRL Press at OxfordUniversity Press (1984, ISBN 0-904147-74-6), which are incorporatedherein by reference. In such embodiments, a nucleoside and/oroligonucleotide hydroxyl-containing compound is immobilized on a solidsupport and reacted within an automated DNA Synthesizer with anucleoside phosphitylating agent in the presence of a phosphitylationactivator to form an oligonucleotide. A specified number and sequence ofphosphitylation reactions may be conducted to produce oligonucleotidescomprising different lengths and sequences of nucleosides according tothe present invention.

[0057] Any suitable solid support materials may be adapted for use inthe present invention. Examples of suitable solid support materialsinclude controlled-pore glass (“CPG”), polystyrene, silica, cellulosepaper, and combinations of two or more thereof. A preferred class ofsolid support material includes controlled-pore glass, polystyrene, andcombinations thereof.

[0058] The solid support for use in the present methods may have poresof any suitable size. As will be recognized by those of skill in theart, the choice of pore size depends at least in part upon the size ofthe oligomer to be produced and the nucleotide synthesis procedure used.In light of the teachings herein, those of skill in the art will bereadily able to select a solid support material of appropriate pore sizefor use in a wide variety of applications.

[0059] A variety of solid-support immobilized nucleosides are availablecommerically. For example, a number of n-protected deoxynucleosidesimmobilized on CPG (including 0.2 micromolar Benzoyl-protecteddeoxycytosine on 1000 angstrom CPG) are available from AppliedBiosystems (ABI).

[0060] Any of a wide range of Automated DNA/RNA Synthesizers can beadapted for use in the present invention. Examples of suitable DNASynthesizers include the Model Nos. 3900, 380, 380B, 392 and 394,Expedite 8800, 8905, 8909, Gene Assembler, OligoPilot, OligoPilot II,AKTAoligopilot 10, and AKTAoligopilot 100 available from AppliedBiosystems, as well as, Beckmann Oligo 1000 and 1000M, the MWG BiotechOligo 2000, PolyPlex GeneMachine, Illumina Oligator, MerMade I and II,Imntelligent BioInstruments Primer Station 960, Proligo Polygen,Syntower, and the like. A preferred class of Synthesizer includes Model394, and the like.

[0061] Any suitable amounts of solid-supported hydroxyl-containingcompound and phosphitylating agent may be used according to the presentautomated methods. In certain preferred embodiments, an excess,preferably a fifty fold excess, of phosphitylating agent is used foreach reaction.

EXAMPLES

[0062] The invention is further described in light of the followingexamples which are intended to be illustrative but not limiting in anymanner.

Examples 1-11

[0063] These Examples illustrate the phosphitylation of severalprotected nucleoside reagents with2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite in the presenceof several activators according to the present invention.

[0064] Eleven phosphitylation reactions (1-11) comprising reacting aprotected nucleoside reagent with2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite in the presenceof an acid-base activator according to the present invention wereconducted, and the product yields of each calculated, as described inthe General Procedure, below. The various combinations of protectednucleoside, activator base, activator acid, solvent, and yield for eachof the 11 reactions are listed in Table 1.

[0065] General Procedure: The activator base (1.1 to 1.2 equivalents) isadded to the solvent and 0.95 to 1.1 equivalents of activator acid issubsequently added thereto at ambient temperature to form the activatorsolution. About 1 equivalent of the protected nucleoside is dissolved inabout 10 equivalents of the solvent in a separate vessel and about 3equivalents of the solvent is then distilled off under reduced pressure.About 1 to 1.2 equivalents of2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite is added to thenucleoside mixture at ambient temperature, and the activator solutionprepared previously is then added to the nucleoside mixture at ambienttemperature with vigorous stirring. After 12 hours, the reaction mixtureis diluted with toluene and washed with water. The organic layer isseparated, dried over sodium sulfate if necessary, and concentratedunder reduced pressure. The yield of the desired amidite is thencalculated using HPLC techniques, that is, the resulting product mixtureis run through an HPLC column using an appropriate eluent, and the areaunder the HPLC peaks used to determine the %yield of product in themixture. TABLE 1 Ex- % ample Nucleoside Base Acid Solvent Yield 1Bz-DMT-dA 2-Picoline TFA Methylacetate 95 2 Bz-DMT-dA Syn-Collidine TFAMethylacetate 89 3 Bz-DMT-dA Hilnig Base TFA Methylacetate 98 4Bz-DMT-dC 2-Picoline TFA Isobutylacetate 91 5 Bz-DMT-dC Syn-CollidineTFA Isobutylacetate 95 6 Bz-DMT-dC Hitnig Base TFA Isobutylacetate 92 7iBu-DMT-dG 2-Picoline TFA THF 67 8 iBu-DMT-dG Himig Base TFA THF 92 9DMT-T 2-Picoline TFA THF 94 10 DMT-T Syn-Colhdine TFA THF 95 11 DMT-THunig Base TFA THF 96

Examples 12-18

[0066] These Examples illustrate the phosphitylation of severalprotected nucleoside reagents with2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite in the presenceof a Hünig's Base-TFA activator according to the present invention.

[0067] Seven nucleoside reagents of the Formula III (below) were reactedwith 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite in thepresence of a Hünig's Base-TFA activator according to the GeneralProcedure for Examples 1-11, and the product yield calculated via one oftwo methods: (1) the resulting product mixture is run through an HPLCcolumn using an appropriate eluent, and the area under the HPLC peaksused to determine the %yield of product in the mixture; or (2) theresulting product is purified on a short silica gel column using amethylacetate/toluene mixture (the concentration depending on theparticular product being purified). The appropriate product fractionsare concentrated under reduced pressure and solvent until anapproximately 50% solution of the desired amidite is obtained. Thissolution is added to about 5 to 25 equivalents of petroleum ether toprecipitate the product which is filtered and washed with petroleumether. The product is then dried, weighed, and the percent yieldcalculated.

[0068] wherein B is a moiety derived from N⁶-benzoyl-Adenine (A(Bz)),N⁴-benzoyl-Cytisine (C(Bz)), N²-isobutyroyl-Guanine (G(iBu)), Thymine(T), or Uracil (U), and X is hydrogen, OTBDMS, or methoxy (OMe). Theparticular X and B variables for each nucleoside and the % yield foreach reaction are shown in Table 2. TABLE 2 Example X B % Yield 12OTBDMS A(Bz) 92 - HPLC 13 OTBDMS C(Bz) 93 - HPLC 14 OMe C(Bz) 96 - HPLC15 H G(iBu) 94 - HPLC 16 OTBDMS G(iBu) 92 - HPLC 17 H T 80 - isolated 18OTBDMS U 85 - HPLC

Example 19

[0069] This example illustrates the phosphitylation ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyAdenosine (Bz-DMT-dA)with diisopropylethyl ammonium trifluoroacetate and2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphor-diamidite according of thepresent invention.

[0070] Diisopropylethylamine 6.4g (49.4 mmol) is dissolved in 20 ml ofdry THF in a reaction vessel. Trifluoroacetic acid 4.9g (43.6 mmol) isadded to the THF mixture at ambient temperature to form an activatorsolution for use in the following reaction step.

[0071] Bz-DMT-dA 30g (45 mmol) is dissolved in 185 ml of dry THF in areaction vessel and 50 ml of the THF is then distilled off under reducedpressure to form a reaction mixture. To the reaction mixture is added14.7g (47.2 mmol) of2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite at ambienttemperature. The activator solution prepared above is then added to thereaction mixture at ambient temperature with vigorous stirring. After 12hours, the reaction mixture is diluted with 80 ml toluene and washedwith 50 ml of water. The organic layer is separated and concentratedunder reduced pressure. The resulting product is purified on a shortsilica gel column using methylacetate/toluene (80/20). The appropriateproduct fractions are concentrated under reduced pressure and solventuntil an approximately 50% solution of5′-O-Dimethoxytrityl-2-deoxyAdenosine-(N⁶-benzoyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite(PAm-Bz-DMT-dA) is obtained. The approximately 50% solution is added,with vigorous stirring (approximately 500-600 rpm), to a 1-L stainlesssteel reactor equipped with a mechanical stirrer and containing 500 mlhexane at ambient temperature. After 3 hours the resulting precipitateis filtered, washed with 50 ml hexane and dried yielding 32 g (83%)Pam-Bz-DMT-dA.

Example 20

[0072] This example illustrates the phosphitylation ofN4-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyCytidine (Bz-DMT-dC) withdiisopropylethyl ammonium trifluoroacetate and2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite according of thepresent invention.

[0073] Diisopropylethylamine 22.4g (173 mmol) is dissolved in 30 ml ofdry THF in a reaction vessel. Trifluoroacetic acid 18.4g (164 mmol) isadded to the THF mixture at ambient temperature to form an activatorsolution for use in the following reaction step.

[0074] Bz-DMT-dC 103g (158.3 mmol) is dissolved in 450 ml of dry toluenein a reaction vessel and 100 ml of the toluene is then distilled offunder reduced pressure to form a reaction mixture. To the reactionmixture is added 51.4g (170.5 mmol) of2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite at ambienttemperature. The activator solution prepared above is then added to thereaction mixture at ambient temperature with vigorous stirring. After 12hours, the reaction mixture is washed twice with 100 ml of aqueousammonium acetate solution. The organic layer is separated andconcentrated under reduced pressure. The resulting product is purifiedon a short silica gel column using methylacetate/toluene/triethylamine(100/30/2). The appropriate product fractions are concentrated underreduced pressure and solvent until an approximately 50% solution of5′-O-Dimethoxytrityl-2′deoxyCytidine-(N4-benzoyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite(PAm-Bz-DMT-dC) in toluene is obtained. Using a 3-L stainless steelreactor with mechanical stirrer this solution is added to a solution of19 g of triethylamine in 1880 ml hexane with vigorous stirring (500-600rpm) at 5° C. After 3 hours the resulting precipitate is filtered,washed with 100 ml hexane and dried yielding 112 g (85%) PAm-Bz-DMT-dC.

Comparative Examples 1-3

[0075] Three comparative phosphitylation reactions (C1-C3) comprisingreacting a protected nucleoside reagent with2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite in the presenceof an pyridine-TFA activator were conducted, and the product yields ofeach calculated, according to the General Procedure described above forExamples 12-18. The various combinations of protected nucleoside,solvent, and yield for each of the 3 reactions are listed in Table 3. Asillustrated by the yields in Table 3 (as compared to those of Tables 1and 2), the yields associated with the methods of the present inventionsurprisingly tend to be at least as good, and in many embodiments,better, than those associated with comparable reactions usingconventional activators comprising significantly less-hindered salts ofunsubstituted pyridine. TABLE 3 Example Nucleoside Solvent % Yield C1Bz-DMT-dA Methylacetate 90-isolated C2 Bz-DMT-dC Isobutylacetate91-isolated C3 DMT-T THF 95-isolated

Example 21

[0076] This Example illustrates the production of two oligonucleotidesequences (5′-ACGATGATGTTCTCGGGCTTC-3′) and (5′-TTTTTTTTTTC-3′) usingautomated synthetic techniques according to the present invention.

[0077] An ABI 394 DNA Synthesizer was equipped with 4 synthesis columnscomprising 0.2 micromole benzoyl-protected deoxycytosine on CPG (1000angstroms) (from ABI). The Synthesizer was further equipped with 4bottles, each comprising one of four 3′-O-phosphoramidites (based on dC,dA, dG, and T, respectively) to act as the sources of nucleosidephosphitylating agents in the reaction. The Synthesizer was furtherequipped with sources of the following solutions:

[0078] Activator solution: A 1.0M Hunig's base/TFA complex (produced bycombining 8.9 grams of diisopropyethylamine (Aldrich Biotech) with 8.9grams of TFA in 43.0 grams acetonitrile (Honeywell Burdick andJackson)); Deblock - T:  3% trichloroacetic acid in dichloromethane; ACap: 10% acetic anhydride/10% pyridine/80% THF; B Cap: 10%N-methylimidazole/80% THF; and Oxidation T: 0.02 M iodine/2% water/20%pyridine/78% THF

[0079] The nucleoside phosphitylation agents were reacted in sequence,in a fifty-fold excess per coupling, to achieve the desiredoligonucleotides “fos-21” (5′-ACGATGATGTTCTCGGGCTTC-3′) and “CT10”(5′-TTTTTTTTTTC-3′). HPLC analysis was performed using an Agilent 1100Series HPLC equipped with a PDA detector. Agilent ChemStation for LC 3Dsoftware was used to collect and analyze the data. The hplc column usedwas a Dionex DNAPak 100 (4×250 mm) column. A linear gradient with a 1.0ml/min flow rate was used. The mobile phase was: B, 10 mM NaClO₄, 10 mMTris-Cl pH, 8.3; D, 300 mM NaClO₄, 10 mM Tris pH, 8.3. The gradientprogram was as follows: Oligonucleotide HPLC Analysis Gradient Program -45 min Time (min) % B % D 0 100 0 5 75 25 30 40 60 35 0 100 40 100 0

[0080] The oligonucleotides were prepared for analysis at aconcentration of 50 μg per ml in water. The OD260 peak crude DNA yieldvalues were used to calculate the concentrations. Sample injections of30 μl were made. Four samples for each oligonucleotide were tested. Theaverage yields were measured to be about 42.7% for fos -21 and about85.2% for CT-10.

Comparative Example 4

[0081] This Example illustrates the synthesis of two oligonucleotidesequences (5′-ACGATGATGTTCTCGGGCTTC-3′) and (5′-TTTTTTTTTTC-3′) usingtetrazole and pyridine-TFA activators in automated synthesis.

[0082] The oligonucleotide sequences (5′-ACGATGATGTTCTCGGGCTTC-3′) and(5′-TTTTTTTTTTC-3′) were each synthesized and tested as described inExample 22, except conventional activator solutions were used. In oneexperiment, both (5′-TGATGTTCTCGGGCTTC-3′) and (5′-TTTTTTTTTTC-3′) wereproduced using tetrazole from Honeywell Burdick and Jackson, Inc. as anactivator. In another experiment, (5′-ACGATGATGTTCTCGGGCTTC-3′) and(5′-TTTTTTTTTTC-3′) were both produced using a pyridine-TFA activator asdescribed in U.S. Pat. No. 6,274,725.

[0083] The average yield of (5′-ACGATGATGTTCTCGGGCTTC-3′) usingtetrazole as an activator was 65.6% and using pyridine-TFA was 58.1%.The average yield of(5′-TTTTTTTTTTC-3′) using tetrazole as an activatorwas 87.3% and using pyridine-TFA was 86.0%.

What is claimed is:
 1. A method of producing a phosphitylated compoundcomprising the step of reacting a hydroxyl-containing compound with aphosphitylating agent in the presence of a phosphitylation activatorselected from the group consisting of: (1) acid-base complexes derivedfrom an amine base of Formula I

wherein R, R¹, and R² are independently C₁-C₁₀ alkyl, C₁-C₁₀ cycloalkyl,C₁-C₁₀ aryl, C₁-C₁₀ aralkyl, C₁-C₁₀ heteroalkyl, or C₁-C₁₀ heteroaryl;(2) acid-base complexes derived from an amine base of Formula II

wherein R³, R⁴, R⁵, R⁶, and R⁷ are independently hydrogen, C₁-C₁₀ alkyl,C₁-C₁₀ cycloalkyl, C₁-C₁₀ aryl, C₁-C₁₀ aralkyl, C₁-C₁₀ heteroalkyl, orC₁-C₁₀ heteroaryl, and at least one of said R³, R⁴, R⁵, R⁶, and R⁷ isnot hydrogen.; (3) acid-base complexes derived from a diazabicyclo aminebase; (4) zwitterionic amine complexes; and (5) combinations of two ormore thereof, to produce a phosphitylated compound.
 2. The method ofclaim 1 wherein said phosphitylation activator is an acid-base complexderived from an amine base of Formula I.
 3. The method of claim 2wherein said amine base of Formula I is selected from the groupconsisting of diisopropylethylamine, tripropylamine, triethylamine,trimethylamine, diethylmethylamine, NMM, TMEDA, tribenzylamine, andcombinations of two or more thereof.
 4. The method of claim 3 whereinsaid amine base of Formula I is diisopropylethylamine.
 5. The method ofclaim 2 wherein said phosphitylation activator is further derived froman acid selected from the group consisting of trifluoroacetic acid,dichloroacetic acid, methane sulfonic acid, trifluoromethane sulfonicacid, 4-pyridiniumethylene sulfonic acid, non-aqueous hydrogen chloride,non-aqueous hydrogen bromide, non-aqueous hydrogen iodide, and HBF₄. 6.The method of claim 4 wherein said acid-base complex is further derivedfrom trifluoroacetic acid.
 7. The method of claim 1 wherein saidphosphitylation activator is an acid-base complex derived from an aminebase of Formula II.
 8. The method of claim 7 wherein said amine base ofFormula II is selected from the group consisting of dimethylaniline,DMAP, 4-dimethylaminopyridium, methylpyridine, 2-picoline, 3-picoline,dimethylpyridine, 2,6-lutidine, trimethylpyridine, 2,4,6-collidine,syn-collidine, tetramethylpyridine, pentamethylpyridine, andcombinations of two or more thereof.
 9. The method of claim 8 whereinsaid amine base of Formula II is 2-picoline or syn-collidine.
 10. Themethod of claim 8 wherein said phosphitylation activator is furtherderived from an acid selected from the group consisting oftrifluoroacetic acid, dichloroacetic acid, methane sulfonic acid,trifluoromethane sulfonic acid, 4-pyridiniumethylene sulfonic acid,non-aqueous hydrogen chloride, non-aqueous hydrogen bromide, non-aqueoushydrogen iodide, and HBF₄.
 11. The method of claim 9 wherein saidacid-base complex is further derived from trifluoroacetic acid.
 12. Themethod of claim 1 wherein said phosphitylation activator is an acid-basecomplex derived from a diazabicyclo amine base selected from the groupconsisting of DBU, DBN, 1,1,3,3-tetramethylguanidine and combinationsthereof.
 13. The method of claim 1 wherein said phosphitylationactivator is a zwitterionic amine complex.
 14. The method of claim 13wherein said zwitterionic amine complex comprises pyridineethansulfonicacid.
 15. The method of claim 1 wherein said hydroxyl-containingcompound is a nucleoside or an oligomer derived therefrom.
 16. Themethod of claim 15 wherein said nucleoside is a 5′-O-protectednucleoside.
 17. The method of claim 1 wherein said phosphitylating agentis selected from the group consisting ofbis-diisopropylamino-2-cyanoethoxyphosphine;dialkoxy(dialkylamino)phosphines; alkoxy-alkyl(dialkylamino)phosphines,bis(N,N-diisopropylamino)-2-methyltrifluoroacetylaminoethoxyphosphine;bis(N,N-diisopropylamino)-2-diphenyl-methylsilylethoxyphosphine;(allyloxy)bis(N,N-dimethylamino)-phosphine;2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite;methoxy-N,N,N′,N′-tetraisopropylphosphorodiamidite;methyl-N,N,N′,N′-tetraisopropylphosphorodiamidite; and mixtures of twoor more thereof.
 18. The method of claim 17 wherein said phosphitylatingagent is selected from the group consisting of2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite andmethoxy-N,N,N′,N′-tetraisopropylphosphorodiamidite.
 19. The method ofclaim 18 wherein said phosphitylating agent is2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite.
 20. The methodof claim 1 further comprising the step of precipitating thephosphitylated compound in a precipitation solvent.
 21. The method ofclaim 20 wherein said precipitating step comprises providing a compoundsolution comprising a phosphitylated compound and a solvent andcontacting said compound solution with a precipitation solvent toprecipitate the phosphitylated compound.
 22. The method of claim 21wherein said precipitation solvent comprises petroleum ether.
 23. Amethod of producing a phosphitylated compound comprising the step ofreacting a 5′-O-protected-nucleoside with a phosphitylating agent in thepresence of a trifluoroacetic acid-diisopropylethylamine complex to forma 3′-O-phosphoramidite.
 24. The method of claim 23 wherein saidphosphitylating agent is2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite.
 25. The methodof claim 24 wherein said 5′-O-protected-nucleoside is selected from thegroup consisting ofN⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyAdenosine,N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyCytidine,N²-isobutyroyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyGuanosine, and5′-O-(4,4′-dimethoxytrityl)-Thymidine.
 26. The method of claim 23wherein said 3′-O-phosphoramidite is selected from the group consistingof5′-O-Dimethoxytrityl-2′-deoxyAdenosine(N⁶-Benzoyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,5′-O-Dimethoxytrityl-2′-(N⁴-Benzoyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,5′-O-Dimethoxytrityl-2′-deoxyGuanosine(N²-isobutyroyl)-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite,and5′-O-Dimethoxytrityl-thymidine-3′-N,N-diisopropylamino-O-(2-cyanoethyl)phosphoramidite.27. The method of claim 23 wherein said 3′-O-phosphoramidite is purifiedvia precipitation of said 3′-O-phosphoramidite in a precipitationsolvent.
 28. A phosphitylated compound produced according to the processof claim
 23. 29. A method of producing a phosphitylated compoundcomprising the step of reacting an oligomer derived from a5′-O-protected-nucleoside with a 3′-O-phosphoramidite in the presence ofa trifluoroacetic acid-diisopropylethylamine complex to form aphosphitylated oligomer compound.
 30. The method of claim 29 whereinsaid reaction is conducted in an automated DNA synthesizer.
 31. Aphosphitylated oligomer compound produced according to the process ofclaim
 29. 32. A method of purifying a phosphitylated compound comprisingthe steps of providing a compound solution comprising a phosphitylatedcompound and a solvent and contacting said compound solution with aprecipitation solvent to precipitate a purified phosphitylated compound.33. The method of claim 32 wherein said precipitation solvent comprisespetroleum ether.
 34. The method of claim 33 wherein said contacting stepis conducted at a temperature of from about 5° C. to about 25° C. 35.The method of claim 34 wherein said contacting step is conducted in astainless steel vessel.